This invention relates to a hypoallergenic composition containing specific tolerogenic peptides of proteins, wherein the composition can induce oral tolerance to the native proteins. It also relates to the use of tolerogenic peptides of milk protein for the preparation of a composition inducing immunological tolerance to milk proteins.
During the common process of nutrition, dietary proteins are presented to the immune system via the gut, followed by an immune unresponsiveness to the ingested nutrients. This vital phenomenon, called oral tolerance, is efficient for the large majority of people. If oral tolerance fails, food allergy occurs, requiring the strict avoidance of the incriminated food.
Targeted food avoidance represents a hard task for adult patients with food allergy. For example, to strictly remove cow""s milk from the diet of allergic infants might be even more difficult, especially if breastfeeding is not possible or desired.
The allergies to cow""s milk and to the formulas containing cow""s milk adapted to the needs of infants are due to the fact that the proteins of cow""s milk differ from the proteins of mother""s milk and can constitute allergens. Among whey proteins, xcex2-lactoglobulin is the major component and is a strong allergen.
Besides breastfeeding, the primary recommendation for prevention, hypoallergenic formulae are systematically prescribed to xe2x80x9cat riskxe2x80x9d newborns, namely asymptomatic infants with atopic parents.
In contrast to adapted formulae, cow""s milk proteins have been hydrolyzed in hypoallergenic formulae, to decrease the potential allergenicity. This approach has been demonstrated to be efficient in order to prevent sensitization by native proteins present in the adapted formulae.
Thus, in U.S. Pat. No. 4,293,571 a protein hydrolysate was prepared by pancreatic hydrolysis, coagulation of non-hydrolyzed proteins by a heat treatment and ultrafiltration to eliminate the coagulated residual proteins and the macropeptides that could constitute allergens. Also, U.S. Pat. No. 5,039,532 provides an improved process for the preparation of a hydrolysate of animal milk proteins substantially reduced in allergens, in which a whey product was subjected to enzymatic hydrolysis.
Two different types of hypoallergenic formulae are proposed to high-risk babies: partially and extensively hydrolyzed formulae, differentiated by the extent of hydrolysis of the native proteins. But as is clear from the art cited above, the primary focus to date in dealing with allergies to cow""s milk has been to find preparations that will not induce an allergic response, i.e. to provide non-allergenic formulations. Nonetheless, while such formulations have permitted a person allergic to cow""s milk to avoid an allergic response, they do not solve the problem which is to enable a person to drink unaltered milk products.
More recently, it has been investigated that partially hydrolyzed milk formulations are not only of reduced allergenicity but can induce immunological tolerance to milk proteins (see, e.g., European patent application 0 827 679). Extensively hydrolyzed formulae are specially designed for treating patients allergic to cow""s milk proteins but their ability to induced long-term oral tolerance is questioned.
Thus, European patent application 0 629 350 discloses the use of non-allergenic whey protein hydrolysates which are said to be capable of inducing cow""s milk protein tolerance. Although this patent application indicates that whey protein hydrolysates substantially free of allergenic proteins could be used to induce cow""s milk protein tolerance in children at risk of cow""s milk allergy, the present inventors found on analyzing other non-allergenic whey protein hydrolysates that non-allergenicity did not necessarily translate into the ability to induce cow""s milk protein tolerance. Indeed, some of the formulations exhibiting the highest degree of non-allergenicity were found to be unsuitable for inducing cow""s milk protein tolerance.
Although Lo, C. W. and Kleiman, R. E. (American Journal of Clinical Nutrition 1996, 63 (4), 646S-650S) suggest that infant formula containing tolerogenic peptides could be used for treating allergic diseases or suppressing the development of autoimmune disorders, it is clear that the art lacked the information to provide formulae that would be the most suitable for tolerance induction The present invention now overcomes the shortcomings of the art.
This invention relates to a hypoallergenic composition for the induction of protein tolerance in at risk individuals of protein allergy. The composition contains (i) a xe2x80x9cnon-allergenicxe2x80x9d extensively hydrolyzed proteins basis and/or (ii) a free amino acid basis, and comprises as the active ingredient at least one tolerogenic peptide of the allergenic protein.
In a preferred embodiment, the tolerogenic peptide is present in the form of (i) one or more isolated tolerogenic peptidic fractions of hydrolysis of proteinaceous material containing the allergenic protein, and/or (ii) one or more synthetically prepared tolerogenic peptides.
This composition also contains a source of nitrogen which may provide 7 to 25% of the total energy, a source of carbohydrates which may provide at least 28 to 66% of the total energy, a source of lipids which may provide at least 25 to 60% of the total energy and at least one tolerogenic peptide of the different proteins.
A major advantage of this composition is to induce oral tolerance in xe2x80x9cat riskxe2x80x9d individuals, in order to avoid eventual sensitization by use of native tolerogens. Moreover, the tolerogenic peptides derived from protein hydrolysis offer both hypoallergenic and tolerogenic properties and induce oral tolerance at the humoral and cellular levels.
This composition is particularly intended for individuals at risk of milk protein allergy.
Another aspect of the present invention is the use of tolerogenic peptides of milk proteins for the preparation of a hypoallergenic composition intended for mammals susceptible to cow""s milk allergy.
In a preferred embodiment, tolerogenic peptides are from milk origin and particularly from xcex2-Lactoglobulin (xcex2-LG), xcex1-lactalbumin, bovin serum albumin or casein origin.
For the preparation of the composition, tolerogenic peptides may be used in the form of peptidic fractions containing the following peptides: H2N-I-D-A-L-N-E-N-K-COOH, H2N-V-L-V-L-D-T-D-Y-K,-K-COOH or H2N-T-P-E-V-D-D-E-A-L-E-K-F-D-K-COOH from xcex2-Lactoglobulin.
In another aspect, the invention provides a method for the preparation of tolerogenic peptides useful in the induction of protein tolerance in at risk individuals to protein allergy, wherein:
(i) a proteinaceous material containing the allergenic protein is hydrolyzed to a degree of hydrolysis of about 10 to 50%;
(ii) then treated to inactivate residual enzyme activity; and
(iii) the protein hydrolysate solution is clarified and submitted to precipitation treatment or passed into a chromatography column filled with appropriated resin to extract the tolerogenic peptidic fractions.
In the present invention, the term tolerance is to be understood as a state of specific immunological unresponsiveness. Both humoral (antibodies) and cell-mediated (lymphocyte . . . ) pathways of the immune response may be suppressed by tolerance induction. A breakdown of oral tolerance is considered to be the underlying cause of food allergy.
The term xe2x80x9callergenxe2x80x9d is to be understood as a protein or macropeptide capable of initiating allergic reactions in humans, particularly at risk infants or nurslings. Infants are considered being xe2x80x9cat riskxe2x80x9d of protein allergy when either one or two parents or one sibling is atopic.
The term xe2x80x9ctolerogenic peptidexe2x80x9d is to be understood as a proteic fragment or fragments corresponding to parts of the native protein, sized from 200 to 6000 Daltons (xe2x80x9cDaxe2x80x9d), corresponding to about 3 to 50 amino acids, and preferably between 500 to 3000 Da and being able to induce specific oral tolerance to native proteins.
The term xe2x80x9cnon-allergenic basisxe2x80x9d is to be understood as a nitrogen source containing a well-balanced amino-acids composition. The xe2x80x9cnon-allergenicityxe2x80x9d is defined for milk proteins as residual allergenicity of individual whey proteins not exceeding 1 ppm and as residual allergenicity of total caseins not exceeding 10 ppm.
The hypoallergenic composition may contain as a source of nitrogens, peptides or free amino acids and particularly from milk proteins such as whey proteins, xcex1-lactalbumin, xcex2-lactoglobulin, bovine serum albumin, casein acid, caseinates, or xcex1, xcex2, xcexa-casein, for example. The source of nitrogen can provide at least 7 to 25% of the total energy.
As a source of carbohydrates, lactose, saccharose, starch or maltodextrin may be used. Carbohydrates may provide at least 28 to 66% of the total energy.
Vegetable oils or butter oil are preferably used as a source of lipids that may provide at least 25 to 60% of the total energy.
Vitamins, oligoelements and minerals can be added in an amount sufficient to meet daily requirements.
The composition according to the present invention comprises as the active ingredient at least one tolerogenic peptide of the allergenic protein, wherein the tolerogenic peptide has been selected for its ability to induce oral tolerance.
The tolerogenic peptides can be obtained by enzymatic hydrolysis of proteinaceous material containing the allergenic proteins that are responsible for allergies in at risk individuals, followed by isolation of tolerogenic peptidic fractions. These peptidic fractions enriched in said tolerogenic peptides can be obtained by separation of the protein hydrolysate. The tolerogenic peptides may also be present in the composition in the form of synthetically prepared tolerogenic peptides.
The composition contain an amount sufficient to induce oral tolerance which is preferably the one which allows a complete oral tolerance induction, namely the one which prevents from any reaction after DBPCFC (double blind placebo controlled food challenge) performed with cow""s milk. Accordingly, tolerogenic peptides may be present in an amount of about 0.01% to 10% (nitrogen source of the protein), for example and preferably about 0.1 to 0.2% of total peptides.
In the particular case of tolerance to milk proteins, the composition may contain tolerogenic peptides from milk origin such asxe2x80x94lactoglobulin or caseins, for example. Tolerogenic peptides may thus be in the form of a peptidic fraction comprising at least one of the following peptides: H2N-I-D-A-L-N-E-N-K-COOH, H2N-V-L-V-L-D-T-D-Y-K,-K-COOH or H2N-T-P-E-V-D-D-E-A-L-E-K-F-D-K-COOH from-xcex2-lactoglobulin.
In a preferred embodiment, a method for the preparation of tolerogenic peptides comprises the following steps:
(i) a proteinaceous material containing the allergenic protein is hydrolyzed to a degree of hydrolysis of about 10 to 50%;
(ii) then treated to inactivate residual enzyme activity; and
(iii) the protein hydrolysate solution is clarified and submitted to precipitation treatment or passed into a chromatography column filled with appropriated resin and tolerogenic peptidic fractions are recovered.
This preferred method is well suited to treatment of hydrolysates prepared from various protein concentration (Ntot %=N * 6.38) for modifying the ratio of tolerogenic activity by residual antigenicity from proteinaceous material. If one defines arbitrarily the antigenicity of a native protein to be 106 (as 106 xcexcg/g of protein), and the tolerogenic response to be 1, then, for a native protein, this ratio is 10xe2x88x926. Therefore, the ratio qualifying the tolerogenic activity of one given fraction or tolerogenic peptide should be at least 2xc3x9710xe2x88x922.
The proteinaceous material to be treated may be any composition containing protein material and in particular solution or dispersion of milk proteins: whey proteins, acid whey protein, sweet whey proteins, whey protein concentrates, whey protein isolate, demineralized whey powder or caseinates, for example.
In general, the protein content may vary within the range of about 70 to 95% by weight but the starting material is preferably as rich in protein as possible.
The proteins present in the proteinaceous material can be modified with proteolytic enzymes into protein hydrolysate having a degree of hydrolysis (xcex1-amino-N/Ntot) of preferably about 10-50%.
The proteolytic enzymes may be for example, from animal or vegetable origins (pepsin, chymotrypsin, trypsin, intestinal mucosa extract, pancreatic extracts, chymosin, papain, bromelain, ficin), bacterial or fungi origins (serine and metalloproteases from Bacillus subtilis, Bacillus licheniformis, Aspergillus orysae, Aspergillus wentii and acidic proteases from Aspergillus orizae, Aspergillus wentii, Mucor miehei, Mucorpusillus, Endothia parasitica) or a combination of these.
During hydrolysis, concentration of proteinaceous material in solution or in suspension is preferably around 5-20% by weight and could be pasteurized before introducing proteases. The ratio enzyme/protein may be 0.1-10% weight/weight and preferably of about 0.25 to 4%.
Hydrolysis may be conducted at a temperature of about 35xc2x0 C. to 65xc2x0 C., during 30 minutes to 10 hours, preferably 30 min to 4 hours at pH values within the range 2.5 to 11, preferably 4.5, 7.0, 8.0, and 8.5. If desired the pH of the solution can be adjusted and regulated with citric acid, food grade HCl or NaOH, NH4OH, KOH, Ca (OH)2 for instance at a concentration of 2N pure or in blend.
Then, the protein hydrolysate may be submitted to a heat treatment of about 0.1 to 10 min at a temperature of about 70 to 110xc2x0 C. to inactivate residual enzymes (i.e., proteases).
The protein hydrolysate solution thus obtained can be clarified by centrifugation and/or ultrafiltration to remove insoluble and intact proteins respectively, and the clear solution is recovered. It is possible to use at industrial scale different type of membranes (spiral, tubular, flat, allow fibers) made with different materials (minerals, polysulfone, . . . ) and having different cut off limits between 1,000 and 100,000 Daltons. Depending on the type of enzyme, the hydrolysis conditions and the type of membranes the modification of the tolerogenic fractions should be sufficient at this step.
The recovered clear hydrolysate solution can, if desired, be concentrated by evaporation to a dry solid content of 10-50% for a subsequent treatment or spray dried if enrichment in tolerogenic peptides is sufficient.
The protein hydrolysate solution thus obtained can be submitted to precipitation treatment by solvent, acid, or salts, for example, followed by a centrifugation. In the precipitation treatment, concentration of hydrolysate solution increases the yield and reduces the quantities of solvent. For example, ethanol may be added to obtain a final concentration within 15-60% volume/volume at a temperature of about 4xc2x0 C. to 25xc2x0 C. After one hour of incubation, a centrifugation (30 min at 4500 g) should separate soluble and insoluble peptides. Depending on the proteolysate, an acid (phosphoric or hydrochloric, for example) or phospho-calcic precipitation can be used. Then, solvents can be removed by evaporation and salts by electrodialysis.
The clear solution and the insoluble fraction are preferably recovered.
The protein hydrolysate solution thus obtained may be passed into a column filled with adsorption, ion exchange or hydrophobic resin at a flow rate of 0.1-4 column volumes per hour at a temperature of about 4xc2x0 C. to 60xc2x0 C. Before the chromatography treatment, the protein hydrolysate can be concentrated to provide a solution having a dry solid content of 8-35% by weight.
During chromatography, a fraction of peptide is absorbed into the resin by passing the hydrolysate solution into a column filled with the convenient support at a rate of 0.1-4 column volumes per hour. It is possible to use at industrial scale the different types of chromatography as: ion exchange, hydrophobic interactions, reverse phases, adsorption (hydroxyapatite, active charcoal, polystyrene base hydrophobic resins . . . ) or covalent chromatography.
In the chromatography treatment, the amount of hydrolysate solution per liter of resin filled column can be as high as 5 liters with the respect to dry solids of 10%. Preferably, a hydrolysate solution having 20-1000 g of dry solid per liter of resin is passed into the resin filled column. The chromatography treatment may be carried out at a pH of about 2 to 10 preferably 6-8, for the clarified hydrolysate solution. The chromatography treatment can be conducted at a temperature of about 4xc2x0 C. to 60xc2x0 C.
For example, the chromatography treatment to select tolerogenic fractions from xcex2-lactoglobulin may consist in using:
a strong cationic resin equilibrated with 0.1 N HCl at a flow rate of 1 volume/hour. The non-retained fraction was eluted with 3 volumes of water, the second fraction (fraction containing tolerogenic peptides) was eluted with 0-0.5 N NaOH, and the third fraction was eluted with 0.1 N HCl.
a reverse phase (C18) resin equilibrated with pure water. Non retained fraction was eluted with water, then step by step (20% and 40% of ethanol) the second and the third were recovered.
a strong anionic resin equilibrated with 0.1N NaOH. Non retained fraction was eluted by 3 volumes of water. The second fraction was eluted with 0.5N HCl, the third one with 0.1 N NaOH.
The most preferred method is to treat with resin a neutral solution, in that case, no pH adjustment is required-after hydrolysis step and the salt content of the product will be lower.
To conclude the chromatography treatment, the column can be eluted with pure water, then water containing salts, buffer, acids, bases, or organic solvents at a temperature of 4-60xc2x0 C. Elution is realized step by step or by a gradient of concentration. The solutions that have passed through the column are recovered. If necessary, salts, solvents, acids, bases, are removed from the recovered solution, and the recovered solutions can be concentrated to a dry solids content of 35-65% and spray dried.
These peptides are then specific fragments corresponding to a part of the native protein sequence or to a part of the specific tryptic peptides of hydrolyzed protein.
These tolerogenic peptides can be used for the preparation of a composition inducing oral tolerance to native proteins, said composition is intended for mammals susceptible to protein allergy and particularly human and pets.